The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.
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Method Article
Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility
In This Article
Summary
Abstract
Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.
Introduction
Drosophila ORNs respond to a vast number of odorants, with widely ranging carbon chain lengths and a variety of functional groups, including esters, alcohols, ketones, lactones, aldehydes, terpenes, organic acids, amines, sulfur compounds, heterocyclics, and aromatics2,3. Odorants varied in their physicochemical features can have markedly different volatilities, indicated by the vapor pressure of the compound. Notably, biologically relevant odorants for Drosophila melanogaster differ tremendously in their volatility. For example, Ir92a ORNs respond to ammonia4, which i....
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Protocol
1. Preparation of the Hardware for at4 Recording
- Use a pipette puller instrument to prepare electrodes with aluminosilicate glass capillaries (O.D 1.0 mm, I.D. 0.64 mm). Blunt the tip of the reference electrode slightly with a pair of fine forceps to facilitate insertion into the clypeus of the fly (i.e. a rounded plate at the front of the fly head, above the mouthparts).
NOTE: 7-day-old WT males (Berlin) were used in this study. Use AHL saline solution14 as the electrolyte for both electrodes. - Prepare 1 L of AHL by mixing 900 mL of distilled water with 6.312 g of NaCl, 0.373 g of KCl, 0.337 g of NaHCO....
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Results
Our technique was successfully applied to determine the relative efficacy of the trans (Figure 5A) versus cis (Figure 5B) isomers of palmitoleic acid. Our representative data demonstrates that trans-palmitoleic acid is a more effective ligand for Or47b ORNs when compared to the cis isoform (Figure 5C). A single neuron was recorded from each fly, with twelve flies r.......
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Discussion
Here, we described a procedure by which the responses of Or47b ORNs to palmitoleic acid can be robustly induced and recorded. We modified a conventional long-distance odor delivery method2,7,10 to troubleshoot the problem of insufficient pheromone odorant delivery. We addressed the issue of low odorant volatility by delivering the compound via odorant cartridges, the opening of which are positioned within millimeters of the prep.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Ye Zhang for the help with the sample traces and Tin Ki Tsang for the help with the pictures. This work was supported by a Ray Thomas Edwards Foundation Early Career Award and an NIH grant (R01DC015519) to C.-Y.S. and NIH grants (R01DC009597 and R01DK092640) to J.W.W.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Prep Setup & Miscellaneous Materials | |||
| Pipette Puller Instrument | Sutter Instruments Novato CA USA | P97 | Pipette Puller |
| Borosilicate Glass Capillaries | World Precision Instruments Sarasota FL USA | 1B100F-4 | to make holding rods |
| Aluminosilicate Glass Capillaries | Sutter Instruments Novato CA USA | AF100-64-10 | to make electrodes |
| Superfrost Microscope Slides | Fisher Scientific Pittsburgh PA USA | 12-550-143 | for fly-prep station |
| Permanent Double Sided Tape | Scotch St. Paul MN USA | NA | for fly-prep station |
| Upright microscope | Olympus Shinjuku Tokyo Japan | BX51 | for recording rig |
| Plastalina modeling clay | Van Aken North Charleston SC USA | B0019QZMQQ | for prep station and to stablize the holding rod |
| Rapid-Flow Sterile Disposable Filter Unit with SFCA Membrane, 0.45 mm | Nalgene Rochester NY USA | #156-4045 | to sterilize AHL solution |
| Name | Company | Catalog Number | Comments |
| Cartridge Materials | |||
| 200 µL pipette tip | VWR Radnor PA USA | 53508-810 | to make odor cartridges and fly prep |
| Filter Paper | Whatman Maidstone Kent UK | 740-E | to make odor cartridges |
| Vacuum Desiccator | Cole-Parmer Vernon Hills IL USA | VX-06514-30 | to vaporize ethanol solvent |
| Name | Company | Catalog Number | Comments |
| Odorant Materials | |||
| cis-palmitoleic acid | Cayman Chemical Ann Arbor MI USA | #10009871 (CAS # 373-49-9) | Or47b odorant |
| trans-palmitoleic acid | Cayman Chemical Ann Arbor MI USA | #9001798 (CAS # 10030-73-6) | Or47b odorant |
| Ethanol | Spectrum Chemical MFG. New Brunswick NJ USA | E1028-500MLGL | to dilute palmitoleic acid |
| Name | Company | Catalog Number | Comments |
| Rig Setup Materials | |||
| Odorant Cartridge Micromanipulator | Siskiyou Grants Pass OR USA | MX130R | to position the olfactometer |
| Flow Vision software | Alicat Tuscon AZ USA | FLOWVISIONSC | software to control flow rate |
| Mass Controller | Alicat Tuscon AZ USA | MC-2SLPM-D | to control the flow rate for humidified air |
| Mass Controller | Alicat Tuscon AZ USA | MC-500SCCM-D | to control the flow rate for odor stimulation |
| Clampex | Molecular Devices Sunnyvale CA USA | Ver. 10.4 | Data acquisition software |
| Air delivery tube | Ace Glass Vineland NJ USA | 8802-936 | to deliver humidified air |
| 50X objective lens | Olympus Shinjuku Tokyo Japan | LMPLFL50X | recording rig |
| Clampfit 10 | Molecular Devices Sunnyvale CA USA | Ver. 10.4 | software for spike analysis |
| Igor Pro 6 | WaveMetrics Lake Oswego OR USA | Ver. 6.37 | software for data analysis |
| Audio Monitor | ALA Scientific Instruments Farmingdale NY USA | NPIEXB-AUDIS-08B | Aurally reports individual spikes |
| Extracellular Amplifier | ALA Scientific Instruments Farmingdale NY USA | NPIEXT-02F | to increase the amplitude of electrical signals |
| Valve Controller | Warner Instruments | VC-8 | to control the opening of the valve for odor stimulation |
| Recording Electrode Micromanipulator | Sutter Instruments Novato CA USA | MP-285 | to position recording electrode |
| Headstage Amplifier | ALA Scientific Instruments Farmingdale NY USA | EQ-16.0008 | to increase the amplitude of electrical signals |
| Oscilloscope | Tektronix Beaverton OR USA | TDS2000C | Visual report of individual spikes |
References
- Lin, H. -H., et al. Hormonal modulation of pheromone detection enhances male courtship success. Neuron. 90 (6), 1272-1285 (2016).
- Hallem, E. A., Carlson, J. R. Coding of odors by a receptor repertoire. Cell. 125 (1), 143-160 (2006).
- Silbering, A. F., et al.
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